The proposal, entitled "Calcium signaling in vascular smooth muscle", aims to examine in depth the regulation of extracellular Ca2+ entry in the vascular smooth muscle cell. Ca2+entry plays a critical role both in normal and disordered vascular smooth muscle biology. Ca2+ entry is critical for sustained contraction and probably plays a role in growth or differentiation of the vascular smooth muscle cell. In the absence of extracellular Ca2+, or under entry blockade, a contractile response usually cannot be maintained, underlining the importance of the entry system to proper functioning of the vascular smooth muscle cell. Blockade of Ca2+ entry has become an increasingly important tool in the management of hypertension. Greater understanding of this process is needed. Ca2+ entry is part of a two phase system by which intracellular Ca2+ concentration is increased in response to vasoconstrictors and growth factors. In the first phase, Ca2+ is released from intracellular stores, while in the second phase elevated cytoplasmic Ca2+ is maintained by entry of Ca2+ from the extracellular space. While much has been learned about the mechanisms of intracellular Ca2+ release in vascular smooth muscle and other cell types, the mechanisms by which Ca2+ entry is sustained are still poorly defined. We hypothesize that Ca2+ entry is regulated by chemical signals generated on an ongoing basis from the occupied receptor. The signals generated by tyrosine kinase receptors and 7-transmembrane receptors may differ. The goal of this proposal is to show that ligand receptors can induce Ca2+ entry directly via such chemical signals and to determine what the signals are. The specific aims are to determine 1) which products of phosphatidylinositol hydrolysis participate in the signaling of extracellular Ca2+ entry by tyrosine kinase receptors; 2) whether tyrosine kinase receptors sustain Ca2+ entry via alternative signals in addition to the inositol phosphates; 3) whether signaling of Ca2+ entry by tyrosine kinase receptors involves G-proteins; and 4) whether the mechanism for extracellular Ca2+ entry by 7-transmembrane receptors differs from the mechanism used by the tyrosine kinase receptors. The major methods to be used include 1) measurement of intracellular Ca2+ in vascular smooth muscle cells following direct microinjection of the cells with proteins, antibodies, and small molecules; 2) analysis of Ca2+signaling by mutated receptors with altered signaling functions; and 3) analysis of Ca2+ signaling following cotransfection of cells with specific receptor and G protein cDNAs.